Cross‐sectional study on the etiological diagnosis of the patients with chronic prostatitis‐like symptoms by application of the urine‐prostate‐semen test

Abstract Background and Aims The prostatic secretion was considered to be the most important and even only specimen in diagnosis and differential diagnosis of chronic prostatitis like symptoms, but little attention has been paid to other genital organ infections. A urine‐prostate‐semen test (U‐EPS‐S test) was used to investigate the microbial flora of internal genital organs in patients with chronic prostatitis‐like symptoms and their influence on the diagnosis and treatment of the patients. Materials and Methods We randomly selected the patients with chronic prostatitis‐like symptoms for this study and used a U‐EPS‐S test to collect urine, prostatic secretion, and semen specimens. The specimens were inoculated respectively into a suitable culture medium for bacteriological/fungal detection, and the number and distribution of colonies in each isolation culture were observed before and after the therapy. Results All of the specimens from the internal genital organs of these patients were shown as microbe‐positive isolation and the infection rate was 100%. Of these, prostatic secretion with microbe‐positive isolation was obtained in 66 cases (33%), semen with microbe‐positive isolation was obtained in 34 cases (17%), and both prostatic secretion and semen with microbe‐positive isolation were obtained in 100 cases (50%). In the isolates, Gram‐positive microbes were shown as the most common pathogens, accounting for 91.1%. In 200 patients, 95 patients were infected with one microbial species infection, of them 36 were prostatic secretion positive‐isolation (18%), 20 were semen sample positive‐isolation (10%), and 39 were positive‐isolation both prostatic secretion and semen samples (19.5%); 104 patients were infected with two microbial species, of them 30 were prostatic secretion positive‐isolation (15%), 14 were semen sample positive‐isolation (7%), and 60 were positive‐isolation both prostatic secretion and semen samples (30%); one patient was infected with three microbial species and them were isolated from the semen sample (0.5%). In the patients with chronic prostatitis‐like symptoms, the multiple microbial infection (MMI) was accounted for 53.5%, and the multiorgan infection (MOI) was accounted for 67%. Conclusions The U‐EPS‐S test is not only helpful to accurately identify the pathogens and contaminants in the culture isolates, but also the diagnosis and differential diagnosis and also evaluation of the treatment efficacy of the infection in different genital organs. In the patients with chronic prostatitis symptoms, Gram‐positive microbes were the most common causative agents, and MMI and MOI caused by resistant strains of different microbial species have a high incidence.


| INTRODUCTION
The term prostatitis is often used to describe a symptom complex rather than a specific disease entity. The NIH Classification System divided prostatitis into four distinct categories: acute bacterial prostatitis (Category I), chronic bacterial prostatitis (Category II), chronic pelvic pain syndrome (Category III), and asymptomatic inflammatory prostatitis (Category IV). 1 Chronic bacterial prostatitis is one of the most common diseases in andrology; it can also cause some serious complications related to abnormal expression of cytokines and immune responses in the prostate. 2,3 For a long time, diagnosis for the patients was based primarily on clinical manifestations, which are known as chronic prostatitis-like symptoms, digital rectal examination (DRE), cytological, and/or bacteriological tests of urine-prostatic secretion. 4 The Meares-Stamey four-glass urine test and other urine tests are the most commonly used methods in the etiological diagnosis of patients with chronic prostatitis-like symptoms; these tests find the Gram-negative bacteria, especially Enterobacteriaceae spp., to be the most common causative agents. 4,5 However, the treatment of these patients is always unsatisfactory even when an antibiotic is selected according to the results of in-vitro sensitivity tests of the isolation culture of prostatic secretion as most antimicrobials can not diffuse into the prostatic secretion. 6 It also results in the empirical treatments with antimicrobial agents to become a common phenomenon in the therapy of the patients with chronic prostatitis-like symptoms clinically. 7 In recent years, however, it has been confirmed through animal experiments and clinical observations that the dye trypan blue and almost all kinds of antimicrobial drugs can diffuse into the prostatic tissue and prostatic secretion at sufficiently high concentrations. 8,9 Some studies have shown that the prostatic secretions of patients with chronic prostatitis-like symptoms often contain different species of microorganisms or different microbial strains with distinct biological properties or drug sensitivities. 10,11 These suggest that the heterogeneity of biological properties of infecting bacteria, drug resistance of pathogens, and methods of specimen collection might be important factors that affect the treatment efficacy of chronic bacterial prostatitis. A urine-prostate-semen test (U-EPS-S test) was developed and applied in the etiological diagnosis of patients with chronic prostatitis-like symptoms; it has been shown to be helpful in the diagnosis and differential diagnosis and of the infecting pathogens and the infecting organs in the patients. Here, we used the U-EPS-S test to further investigated the microbial flora and their distribution in the genital organs of patients with chronic prostatitislike symptoms and drug sensitivities, and explore the influence of them on the etiological diagnosis and treatment efficacy of the patients.

| Study participants
In total, 200 participants from the clinic services of hospitals in China were randomly selected. All of them were diagnosed with chronic prostatitis according to symptoms and/or the clinical cytological examination of prostatic secretions; however, a bacteriological/fungal test on prostatic secretions was not performed for any.

| The U-EPS-S test
Specimens including segmented urinary stream (IU, TU), expressed prostatic secretion (EPS) and semen (S) were collected respectively by the U-EPS-S test from patients who had stopped the antimicrobial treatment for more than 3 days, the methods were shown in Figure 1.

| Isolation of the pathogens
The specimens (0.1 ml) were inoculated onto blood agar, Sabouraud agar, and mycoplasma agar plates, and incubated under common air or 5% C 2 O conditions at 37°C. 12,13 The pathogens and contaminating microbes were distinguished according to the relative number of each kind of growth on each plate and their distribution in each isolation culture. The term "absolute number" refers to the total number of colonies formed by different microbial species on a medium; "relative number" refers to the total number of colonies formed by one microbial species on a medium and their comparison with the number of the same colonies on another medium; and "distribution" refers to the presence of certain microbes and their number in different culture isolates.

| Identification of the isolates
The organism isolates from the specimens were identified respectively by routine bacteriological/fungal methods and/or gene assays. 13,14 2.5 | Post-therapy pathogen redetection Antimicrobials were selected according to the results of in-vitro sensitivity tests and other relevant necessary factors related to the patient and others, and the dosage and administration (oral or IV) of antimicrobial agents were according to the drug's instructions. In general, the course of treatment was 7-15 days (oral or intravenous administration) for patients with bacterial infection and 20-30 days (oral administration) for those with fungal infections. 9 After the patients finished a course of treatment and had stopped using the antimicrobials for at least 3 days, the IU, TU, RU-EPS, and S were collected again and used for pathogen detection according to the methods described above.

| Statistical analysis
All statistical analyses were performed by SPSS (18.0) software (SPSS Inc.). A nonparametric Wilcoxon's-sign rank test was used for data analysis. p < 0.05 was considered statistically significant. Statistical analysis was performed according to the following formulas.

T T T T
When the size of sample (n) is small: When the rank has many ties:

| General information of the study participants
The urine-prostate-semen test is used for patients with prostatitis-like symptoms. (A) The initial urinary stream (IU) and the third part of the urinary stream (TU) (1-10 ml each) were collected into different sterilized containers and used for microbiological testing. (B) By prostatic massage, the expressed prostatic secretion (EPS) overflowed from the urethral orifice onto a glass slide that was used for the routine test. (C) The EPS within the urethra was collected into a sterilized container by the patient urinating approximately 1-5 ml of residual urine (RU), and a mixture of EPS and RU (RU-EPS) was used for the microbiological testing. (D) Semen collected by masturbation, sexual intercourse, or another method was used for microbiological and routine tests. WANG AND WANG | 3 of 11 years old (6/200, 3%). All of them had been diagnosed with chronic prostatitis based only on their symptoms and/or the cytological examination of prostatic secretions, but the bacteriological/fungal test for their prostatic secretions and semen or even urine never be performed for any. All of the study participants received antimicrobial agent (antibiotics and synthetic antimicrobial drugs) and/or the traditional Chinese medicine empirical treatments respectively. The course of treatment continued on and off for more than 6 months and in some of them even up to 20 years; however, their symptoms were not relieved or they had recurrent attacks.  Figure 2A,B,C,D, and the TU and IU curve of Figure 3A,B,C,D). If the infection was only occurred in patient is prostate, then the absolute number and relative number of colonies in RU EPS would be increased and obvious high than that in TU and semen (such as the cultures of Figure 2A and the curve of Figure 3A). If the infection was occurred in the other internal genital organs but not the prostate, the absolute number and relative number of colonies in semen would be significantly increased and obvious less than that in TU and RU EPS (such as the cultures of Figure 2B and the curve of Figure 3B). If the patient was the infections of prostate and other internal genital organs, the absolute number and relative number of colonies in RU EPS and semen would be significantly increased and obvious higher than that in TU (such as the cultures of Figure 2C and the curve of Figure 3C). If it was only the IU with microbe positive isolation but TU, RU EPS, and semen to be microbe negative isolation, it indicated the prostate and other internal genital organs without infection, it could also be used to indicated that the other genital organ infections of patient have had been cured (such as the cultures of Figure 2D and the curve of Figure 3D).

| Laboratory findings after antimicrobial therapy
After each treatment course, the absolute number (CFU) and species of the pathogens in the IU, TU, RU-EPS, and semen specimens were significantly reduced or completely disappeared compared to the number before therapy, changes in the bacterial species did not occur unless it was severe histopathological damage in the genital organs or the patient was sexually unclean during therapy ( Figure 2D and Table 3). However, the absolute number (CFU) of pathogen in some patients, especially the patients with deferens tract infection after the first course of therapy, would also be showed a significant increase than that before the treatment. In the cases, the pathogens isolated from their specimens were often the drug resistant strains or the patients who did not ejaculate for a long time. Through the drug sensitivity test in-vitro, it was shown that the surviving pathogens or new isolates after each therapy were usually strains that are resistant to the antibiotic just used. It showed a significant reduction in the number of the pathogens in the specimens from most patients after the first course of therapy than that of before the therapy, and the statistical analysis of them showed the p < 0.0001 (Table 4).

| DISCUSSION
The diagnostic value of culture isolates in samples from patients with prostatitis-like symptoms. The part label (A) shows that the absolute number of colonies was relatively large in the IU culture; the colonies consisted of at least three kinds of different bacterial species. In the TU culture, the absolute number of colonies was significantly lower; in the residual urine-expressed prostatic secretion (RU-EPS) culture, the absolute number of colonies was significantly increased. In the RU-EPS culture, at least three kinds of colonies were significantly reduced. In the S culture, only one kind of colony was present. This patient can be diagnosed with multiple microbial infections (MMIs) of the prostate according to the relative number of colonies of each bacterial species and their distribution characteristics in each culture. The part label (B) shows that the absolute number of colonies was relatively large in the IU culture, in which at least two kinds of colonies; that in the TU culture was significantly reduced, with only one kind of colony, and that in the RU-EPS culture was significantly reduced or absent. The absolute number of colonies in the S culture was significantly increased, as were the absolute number and a relative number of colonies, of which the relative number of colonies contained only one kind of colony. This patient can be diagnosed as the infections of deferens tract and/or other internal genital organs but not prostate infection. The part label (C) shows that the absolute number of colonies was relatively large in the IU culture with at least two kinds of colonies, that in the TU culture was significantly reduced, with only one kind of colony, and that in the RU-EPS culture was significantly increased compared to that in the TU culture, with at least two kinds of colonies. The absolute number of colonies in the S culture was also significantly increased, with at least two kinds of colonies diagnosed as both prostate and deferens tract and/or other internal genital organ infection and MMI and multiorgan infection of the internal genital organs. The part label (D) shows that the absolute number of colonies was relatively large in the IU culture, with at least three kinds of colonies; the absolute number and number of colonies in the TU culture, the RU-EPS culture, and the S culture were significantly reduced or absent. This patient can be diagnosed with no infection of the prostate or other internal genital organs; the results can also indicate that the patient has been cured if it is the result of posttherapy pathogen detection. in the TU specimen would be showed as a significant reduction than that of IU specimen or even negative isolation of microbes with the flushing and dilution of the urine stream. It is helpful to identify the isolates in the RU-EPS specimen that came mainly from the prostate but not urine, the microbes in semen specimens came mainly from deferens tract and/or other internal genital organs but not the prostate. Although there were concerns that the volume of the specimens, the time of prostatic secretion and semen collection and the interval between the collection and so on would lead to a reduction in the number of microorganisms in the specimen, we found, in the S-EPS-S test, that the absolute number of the pathogens is variable but the relative number and distribution of them are always characterized. Generally, each course of treatment can decrease significantly the number and species of the drugsensitive strains and even completely eliminate them from the infected organs of patients, but there are some exceptions or special cases. In our previous research 9,12 and this study, it was found that the absolute number of pathogen in some patients, especially in the patients with deferens tract infection undergone first course of the therapy, can also be increased significantly than that before the treatment, they were often the drug-resistant strains. However, this would not affect the trend of the absolute number and species of pathogens to be gradually decreased, and the characteristics of relative number and distribution of them in various specimens.
Through the further standardized treatment, the pathogens in the genital organs of patients will be cleared completely and the patients will also be cured. 9,12 Therefore, the relative number and distribution of the microbes in the specimens are not only helpful for accurate diagnosis and differential diagnosis of the pathogens and the infected organ but also the evaluation of therapeutic effect on etiology.
According to some reports, the Gram-negative bacteria, espe-  numbers, often cause acute or chronic recurrence of prostatitis after 6 months. 16 Animal tests showed that abscesses or other pathological reactions can form within prostatic tissues because of bacterial infection; the pathogens held in pathological tissues can also be released because of extrusion of the prostate gland or the attenuated inflammatory reaction. 15 This seems to explain why the number of pathogens in the RU-EPS or semen specimens of some patients just treated with antimicrobial agents can be higher than that before treatment or why a new pathogen can be found. Therefore, pathogen

| CONCLUSION
The results in this study confirmed: (i) The U-EPS-S test is not only helpful to accurately identify the pathogens and contaminants in the culture isolates but also the diagnosis and differential diagnosis and full access to all of the data in this study and takes complete responsibility for the integrity of the data and the accuracy of the data analysis; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.